Expression And Clinical Significance Of Innate Immune Molecule Toll-like Receptor 4 In Recipient Of Renal Transplantion

In alloimmunity,T lymphocytes are activated by double signals,and then priming the complicated rejection procedure include lymphocytes,cytokins and complements.If antigen present cells(APCs) provide MHC-peptides only,do not present costimulatory signals such as CD80 and CD86 to T lymphocytes at the same time,T lymphocytes will become anergy,immune tolerance will be induced.At present,research for alloimmunity is mainly focused on the immune procedure of T/B lymphocytes and their downstream cytokines.Because of the complexity of the immune procedure after T/B lymphocytes activation,treatments target on one point of the complicated procedure always obtain half the result with twice the effort.Obviously,research focused on APCs-the transmitter of upstream signals for T/B lymphocytes are more superior.The expression of costimulatory molecules on APCs is crucial in determining T-cell immune responses to alloantigens.Decrease of costimulatory molecules such as CD80 and CD86 can converts APCs into tolerogenic cells.These APCs can deprive T lymphocytes the capacity of response to alloantigens.But the question is,who is in charge of the regulatory of CD80 and CD86 expression? How the regulatory happened? In 1997,the discovery of toll like receptor(TLR)-human innate immunity pattern recognition receptor made it possible for the research focused on APCs in alloimmunity.In early time,the research for TLRs was mostly focused on anti-infection immunity.TLRs are'tools' through which innate immunity identify various pathogenic microorganisms.When bind with conservative structure of pathogenic microorganism i.e pathogen-associated molecules,TLRs trigger natural immune response by including the production of inflammatory cytokines,that subsequently signal resulted in the induction of maturation of APCs such as mononuclear/macrophage cells and dendritic cells with up-regulated.Further research implied that TLRs played an important role in alloimmunity and atherogenesis.Among the total,the research of relationship between TLR4 and alloimmunity revealed attracting perspective.Under the help of soluble CD14,TLR4 on APCs binding with LPS,the consequent signals activate NF-kappaB,induce the secretion of proinflammatory cytokine,up-regulation of CD80 and CD86,promote inflammatory process.Research implied,TLR4 may take part in alloimmunity procedure according to analogic pathway,the mechanism is still unclear.At present,research for the role of TLR4 in clinical organ transplantation has just begin,clinical evidence were fewer.A recent clinical study suggests that recipients with a mutant TLR4 genotype(Asp299Gly or The399Ile) manifest reduced lung allograft rejection.Another research shows significantly elevated levels of TLR4 on circulating monocytes in hear-transpant recipients with chronic rejection when compare with control group.Therefore,we investigate the development rule of innate immune molecules TLR4 in clinical renal transplantationand their roles in acute and chronic rejection by immune histochemistry and three-color fluorescent staining flow cytometry.Our study provided clinical and laboratory evidence for the role and clinical application value of TLR4 in organ transplantation,explored the mechanism of TLR4 involved in alloimmunity;We tried our best to offer new ideas for inducing immune tolerance in the future,developing new immunosuppressive drugs aimed at new target,the prevention and treatment of allograft rejection.Chapter 1 Perioperative determination of toll-like receptor 4 and CD80 and their clinical significance in recipients of renal transplantationObjectiveTo investigate the dynamic expression and clinical significance of TLR4/CD80 in patients before and early after renal transplantation,explore the relationship between the expression of TLR4/CD80 and acute rejection.Method32 patients who received renal transplantation in our hospital were involved in our research.All of them were primary recipients,and the level of panal reactive antibody(PRA) less than 1%.The expression of TLR4 and CD80 in CD14 positive monocyte of peripheral blood from patients on the 1^st,4^th,7^th,14^th,21^th,28^th and 35 ^th days after transplantation were measured by three-color fluorescent staining flow cytometry.The patients were divided into rejection group(7 cases) and non-rejection group(25 cases) according to rejection episode record within two weeks.Normal control group(10 cases) was healthy adult volunteers.Diagnosis of acute rejection depended on clinical symptoms,lab test,color doppler sonography and renal biopsy. Results are expressed as mean±SD.A value of P＜0.05 was considered significant. Statistical analysis were performed using SPSS13.0 for windows.TLR4 values were expressed as percentage of positive CD 14+ monocytes.ResultsBefore ransplanstation,the expression of TLR4 and CD80:8.03±0.84%and 0.85±0.31%(n=7) in rejection group,6.14±0.85%and 0.84±0.39%(n=25) in non-rejection group,6.37±0.56%and 0.85±0.35%(n=10) in normal control group. The expression of TLR4 of rejection group was higher than non-rejection group and normal control group(P<0.01).The expression of CD80 of three groups had no significant difference(P=0.995).After ransplanstation,the expression of TLR4 and CD80 in the two groups increased to 16.5±1.02%and 7.82±1.66%in rejection group, 11.6±0.98%and 2.26±0.96%in non-rejection group at the 4^th day,and got to peak at the 7^th day:36.4±4.86%and 9.53±1.97%in rejection group,22.7±3.45%and 1.87±0.72%in non-rejection group,then displayed downtrend,rejection group decreased to 7.10±0.82%and 0.87±0.57%at the 35^th day,non-rejection group decreased to 7.2±0.76%and 0.81±0.37%at the 21^th day.Compared with non-rejection group,rejection group showed higher peak value of expression of TLR4/CD80(P<0.01) and longer consistant time.ConclusionsThe high expression of TLR4 increased the risk of acute rejection.Early after renal transplantation,TLR4 was up-regulated and contributed to the happeness of acute rejection.Chapter 2 Determination of toll-like receptor 4 in acute rejection after renal transplantation.ObjectiveTo investigate the relationship between the expression of TLR4 in CD14 positive monocyte of peripheral blood and acute rejection happened 1 year after transplantation,explore the change of TLR4 after methylprednisolone(MP) pulse therapy. Method13 recipients who developed acute rejection,20 recipients with normal graft function and 20 healthy volunteers were involved in our research.All of the recipients were performed renal transplantation in our hospital at least one year ago.We used flow cytometry to assess TLR4 expression among circulating CD14 positive monocytes before and after methylprednisolone(MP) pulse therapy in recipients with acute rejection.Results are expressed as mean±SD.A value of P＜0.05 was considered significant.Statistical analysis were performed using SPSS13.0 for windows.ResultsWe demonstrated significantly higher TLR 4 expression on circulating monocytes among renal transplantation recipients with acute rejection compared with those in clinically stable with normal renal function and healthy volunteers.MP pulse therapy significantly inhibited the expression of TLR 4 on the monocytes of recipients with acute rejection.The degree of inhibition was related with the reactivity of MP pulse therapy.ConclusionsThe expression of TLR4 in acute rejection after renal transplantation was up-regulated.Monitoring of the level of TLR4 can be used to evaluate the reactivity of MP pulse therapy.Chapter 3 Expression of toll-like receptor 4 in renal allograft tissueObjectiveTo investigate the expression of TLR4 in the renal tissue of recipients with acute rejection and chronic rejection.Method20 recipients who underwent kidney transplantation in our hospital were included in this study:acute-rejection group(n＝10),chronic-rejection group(n＝10). 10 control group were included.We used immune histochemistry combined with Banff 97 classification to investigate the expression of TLR4 in graft tissue.Results are expressed as mean±SD.A value of P＜0.05 was considered significant. Statistical analysis were performed using SPSS13.0 for windows.ResultsThe expression of TLR4 were found in all of three groups'graft tissue. Acute-rejection group and chronic-rejection group were higher than control group. No significance was found in acute-rejection group and chronic-rejection group.In acute-rejection group,the expression of TLR4 were in those patients who had higher classification of pathology.The main source of TLR4 were renal tubular epithelial cells and monocytes.ConclusionsThe expression of TLR4 were increased in renal allograft patients with acute rejection or chronic rejection.